Abstract: Therapeutic proteins are revolutionizing modern medicine, but anti-biotherapeutic immune responses can manifest a range of clinical complications including loss of efficacy, toxicity, or even more serious life-threatening reactions. To enable biotherapeutics to evade the human immune system, we are developing a multi-objective protein design platform that seeks to simultaneously mitigate diverse immunogenicity risk factors while preserving protein structure and function. In the original R01 award, we focused efforts on one key driver of anti-protein immune responses: T cell epitopes, which are immunogenic peptide fragments processed from biotherapeutics and displayed in the context of MHC immune molecules. We were able to comprehensively address design considerations for mutagenic deletion of T cell epitopes within monomeric proteins, and ultimately our computationally-driven approach allowed us to demonstrate, using humanized mice, the direct connection from elimination of T cell epitopes to reduction of anti-drug antibodies to enhancement of therapeutic efficacy. The attached renewal proposal will pursue advances necessary for functional deimmunization of next-generation biotherapies: scaling up from monomeric proteins to more complex structures, addressing MHC immune recognition of intracellular as well as extracellular therapeutic agents, accounting for interrelationships between aggregation and immunogenicity, and simultaneously deleting T cell epitopes and antibody epitopes, where the latter are immunoglobulin binding sites on the surface of intact proteins. The proposed computational algorithms will be engineered to enable design of both individual protein variants and entire combinatorial libraries, using integrated models to assess mutational implications on immunogenicity risk factors and protein structure-function relationships. Fluorescent protein tracers are vital tools in modern biomedical research, yet they manifest the full range of immunogenicity issues described above. Therefore, fluorescent proteins represent a clinically relevant yet experimentally pliable system with which to test and refine our algorithms. While the project aims to produce cutting-edge fluorescent protein tracers, its broader impact is grounded in the universally applicable nature of the proposed deimmunization methods. Ultimately, we anticipate that the sophisticated algorithms emerging from this project will enable development of diverse next-generation biotherapies. For example, future targets might include viral vectors for gene therapy, which must stealthily traverse the extracellular environment in route to intracellular sites of action; aggregation-prone drug candidates, whose aberrant self-association must be surgically remodeled to evade the immune system; and proteins derived from commensal or common pathogenic bacteria, for which human subjects may exhibit preexisting antibody responses.